Direct Cardiac Reprogramming: Advances in Cardiac Regeneration

0 downloads 0 Views 1MB Size Report
Apr 29, 2015 - Because the human heart has minimal ability for endoge- ... cardiac fibroblasts to cardiomyocytes by specific transcrip- tion factor combinations ...
Hindawi Publishing Corporation BioMed Research International Volume 2015, Article ID 580406, 8 pages http://dx.doi.org/10.1155/2015/580406

Review Article Direct Cardiac Reprogramming: Advances in Cardiac Regeneration Olivia Chen and Li Qian Department of Pathology and Laboratory Medicine, McAllister Heart Institute, University of North Carolina, Chapel Hill, NC 27599, USA Correspondence should be addressed to Li Qian; li [email protected] Received 25 March 2015; Accepted 29 April 2015 Academic Editor: Shinsuke Yuasa Copyright © 2015 O. Chen and L. Qian. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Heart disease is one of the lead causes of death worldwide. Many forms of heart disease, including myocardial infarction and pressure-loading cardiomyopathies, result in irreversible cardiomyocyte death. Activated fibroblasts respond to cardiac injury by forming scar tissue, but ultimately this response fails to restore cardiac function. Unfortunately, the human heart has little regenerative ability and long-term outcomes following acute coronary events often include chronic and end-stage heart failure. Building upon years of research aimed at restoring functional cardiomyocytes, recent advances have been made in the direct reprogramming of fibroblasts toward a cardiomyocyte cell fate both in vitro and in vivo. Several experiments show functional improvements in mouse models of myocardial infarction following in situ generation of cardiomyocyte-like cells from endogenous fibroblasts. Though many of these studies are in an early stage, this nascent technology holds promise for future applications in regenerative medicine. In this review, we discuss the history, progress, methods, challenges, and future directions of direct cardiac reprogramming.

1. Introduction Cardiac diseases cause major morbidity and mortality worldwide. The American Heart Association’s yearly review of cardiovascular disease reports that 1 out of every 7 deaths in the United States in 2011 was caused by coronary heart disease [1]. Coronary heart disease predisposes to acute cardiac injuries such as myocardial infarction (MI), which often result in significant and permanent losses of functional cardiomyocytes. Because the human heart has minimal ability for endogenous cardiomyocyte regeneration [2], reparative responses to cardiac cell death primarily rely on activated fibroblasts forming scar tissue. This response to injury fails to restore cardiomyocyte function and often leads to undesirable outcomes including development of arrhythmias and chronic heart failure. Unfortunately, short of heart transplantation, current therapies do not restore declining pump function following cardiac injury. Efforts to address this issue have led to the exploration of several cellular approaches aimed at restoring functional cardiomyocytes; these include stimulating expansion and differentiation of endogenous cardiac progenitor cells and lineage differentiation of embryonic stem cells

or induced pluripotent stem cells. An alternative approach centers on converting nonmyocyte cells directly into new cardiomyocytes (Figure 1). Recently, promising advances have been achieved by directly reprogramming mouse cardiac fibroblasts (CFs) into functional cardiomyocytes in vitro and in vivo by transcription factor expression [3, 4]. Specific combinations of microRNA (miRs) have also been reported to yield similar reprogramming [5]. In human cells, direct reprogramming has also been used to convert dermal and cardiac fibroblasts to cardiomyocytes by specific transcription factor combinations or combinations of transcription factors and miRs [6–8]. Together, these studies describe exciting developments in direct cardiac reprogramming and highlight the potential that cellular therapies hold for future regenerative cardiac therapies. Here, we review the history, recent advances, major challenges, and future directions of direct cardiac reprogramming.

2. Master Genes Regulate Cell Fate In 1986, the Weintraub laboratory [9] laid the groundwork for muscle cell reprogramming by demonstrating that somatic

2

BioMed Research International In vitro Gata4, Mef2c, Tbx5 Gata4, Mef2c, Tbx5, Hand2 Gata4, Mef2c, Tbx5, myocardin, SRF Gata4, Mef2c, Tbx5, myocardin, Nkx2.5 Gata4, Mef2c, Tbx5, Hand2, Nkx2.5, SB432542 Cardiac fibroblasts

Gata4, Mef2c, Tbx5, miR-133

In vitro iCMs

Mef2c, Tbx5, myocardin miR-1, miR-133, miR-208, miR-499, JI1

In vivo Gata4, Mef2c, Tbx5 Gata4, Mef2c, Tbx5, Hand2 Gata4, Mef2c, Tbx5, thymosin 𝛽4

Gata4, Mef2c, Tbx5, VEGF miR-1, miR-133, miR-208, miR-499, JI1 Injured adult heart

In vivo iCMs

Figure 1: Methods of direct cardiac reprogramming. Several approaches for converting mouse or rat fibroblasts to cardiomyocyte-like cells in vitro and in vivo have been reported and are summarized here. In vitro reprogramming predominantly yields partially reprogrammed cells, while in vivo reprogramming yields more mature, fully reprogrammed cardiomyocyte-like cells. iCMs denotes induced cardiomyocytes.

cells could be induced to adopt a skeletal muscle cell fate in vitro, through overexpression of the skeletal muscle master regulator gene, MyoD. Their experiments showed that forced expression of the MyoD gene, which encodes a transcription factor containing a basic helix-loop-helix (bHLH) domain, was capable of inducing expression of several muscle-specific proteins including myosin heavy chain and muscle creatinine kinase [10]. The ability of master regulator genes to govern cell fate changes in vivo was later shown by Halder et al. [11] who ectopically expressed eyeless, the fly homologue of the vertebrate Pax6 gene, in Drosophila and observed ectopic formation of eye structures. Similarly, Santerre et al. [12] showed that transgenic mice overexpressing bmyf, the bovine homolog of the human bHLH domain-containing, myogenic determination transcription factor gene myf5, developed skeletal-muscle like cells in the brain. Together, these studies led to the recognition that in vivo ectopic expression of specific master regulator genes could stimulate cell fate changes. Indeed, in vivo reprogramming was also

achieved by Murry et al. [13] who reported that adenovirus delivery of MyoD to cryoinjured myocardium in rats could cause cardiac fibroblast conversion to skeletal muscle cells. However, successful myogenic conversion, determined by coexpression of MyoD and sarcomere myosin, required high viral doses and occurred in only 14% of MyoD-expressing cells. The lower efficiency of conversion suggests the existence of substantial barriers to the described method of skeletal muscle reprogramming. Nevertheless, these discoveries formed the foundation of direct cellular reprogramming by transcription factor expression and served as an impetus for current advancements in the generation of induced cardiomyocytes.

3. Induced Pluripotency and Direct Cellular Reprogramming In the 1960s, Gurdon [14] challenged the view of permanently fixed cell fates by showing that transfer of a somatic cell

BioMed Research International nucleus into an enucleated frog egg could reprogram the somatic nucleus to a pluripotent state. In 2006, Takahashi and Yamanaka [15] discovered that conversion of somatic cells to pluripotency could be achieved by coexpressing four transcription factors. They successfully reprogrammed mouse fibroblasts to induced pluripotent stem cells (iPSCs) using transcription factors Oct4, Sox2, Klf4, and c-Myc (OSKM). The possibility of changing somatic cells to potentially any other cell lineages sparked many exciting developments in iPSC technology, which are summarized in several other reviews [16, 17]. Advances in iPSC technology also encouraged novel directions in direct cellular reprogramming. Breaking from the MyoD model of single transcription factormediated reprogramming, laboratories worldwide began reporting combinations of transcription factors that could induce different cell fate changes. Efe et al. [18] reported that addition of a small molecule Janus kinase (JAK) inhibitor in mouse embryonic fibroblasts (MEFs) transiently expressing OSKM factors in reprogramming media modified to hinder iPSC induction and promote cardiomyogenesis resulted in formation of spontaneously contracting cardiomyocyte colonies. While there was a minute (1%) population of iPSC-like cells achieved following this method, the authors determined that iPSC and cardiomyocyte generation were parallel processes; that is, reprogramming to cardiac cell fate was unlikely to have passed through a pluripotent intermediate [18]. This study was the first to show cardiomyocyte generation from MEFs by the combination of transcription factor expression, specific culture conditions, and small molecules. The breadth of direct reprogramming applications for generating a variety of cell lineages has also been demonstrated by many other laboratories. Utilizing a specific combination of three transcription factors, Zhou et al. [19] directly reprogrammed exocrine pancreas cells into insulin-secreting pancreatic 𝛽-cells in vivo. Shortly thereafter, direct reprogramming of fibroblasts into other cell lineages generated neurons [20], hepatocytes [21], endothelial cells [22], hematopoietic stem cells [23], and cardiomyocytes [4]. Herein, we will discuss advances specifically related to direct cardiac reprogramming.

4. In Vitro Direct Cardiac Reprogramming With the success of reprogramming to pluripotency achieved through expression of a combination of transcription factors, efforts to identify a master regulator for cardiomyocyte transdifferentiation shifted toward efforts to identify multiple factors that together could activate the cardiac program. In 2010, Ieda et al. [4] identified a combination of three transcription factors, Gata4, Mef2c, and Tbx5 (GMT), that was sufficient to convert mouse cardiac and dermal fibroblasts into functional cardiomyocyte-like cells in vitro. In order to identify the GMT reprogramming factors, the authors created an in vitro assay to determine the minimal number of factors required to convert nonmyocyte cells into a cardiomyocyte cell fate. Transgenic mice were generated to express enhanced green fluorescent protein (EGFP) driven by the 𝛼-myosin heavy chain (𝛼MHC) promoter, which is activated in mature cardiomyocytes and thus permits their identification by

3 green fluorescence. Cardiac fibroblasts were extracted from neonatal transgenic mice and 𝛼MHC-EGFP− /Thy1+ fibroblasts were purified by fluorescence-activated cell sorting (FACS). Fourteen transcription factors of known importance in cardiac development were then retrovirally overexpressed in the purified cardiac fibroblasts. A small number of EGFPpositive cells were observed, suggesting successful cell fate conversion. Serial deletion of individual transcription factors from the original pool of 14 transcription factors yielded the three transcription factors, GMT, which together were capable of inducing EGFP expression in 15–20% of cells, termed induced cardiomyocytes (iCMs). While some iCMs expressed additional cardiac markers like cardiac Troponin T (cTnT) and sarcomeric 𝛼-actinin, assembled sarcomere structures, and demonstrated global enrichment in transcripts similar to the cardiomyocyte transcriptome, the majority of iCMs were likely only partially reprogrammed. Despite this, many iCMs could generate Ca2+ transients and 4 weeks after reprogramming, spontaneous beating was observed in ≈0.5% of 𝛼MHC-EGFP+ /cTnT+ iCMs, which also exhibited action potentials comparable to those of adult ventricular cardiomyocytes [4]. Furthermore, iCMs demonstrated epigenetic shifts toward a neonatal cardiomyocytelike state at the cardiac-specific genes Actn2, Ryr2, and TnnT2, suggesting that reprogramming with the GMT factors induces changes in the epigenetic status of reprogramming cells. While it is possible that the observed iCMs were actually remote cardiac progenitor cells contaminating the fibroblast population, this is unlikely for two reasons. First, use of genetic lineage tracing showed that iCMs did not express cardiac progenitor markers (i.e., Mesp1 and Isl1) during reprogramming. Second, the GMT factors also successfully reprogrammed 15% of neonatal tail-tip fibroblasts (TTF) to 𝛼MHC-EGFP+ iCMs, which also showed expression of cTnT and generation of Ca2+ transients; these TTF-derived iCMs, however, did not beat and the efficiency of their generation was reduced. While these results demonstrate exciting developments in direct cardiac reprogramming, the efficiency of in vitro direct reprogramming by this method is low (50%) in the human heart [38], a clinically important step for the direct cardiac reprogramming field will be to translate reprogramming technology to human fibroblasts. Nam et al. [6] reported successful induction of cTnT expression in human fibroblasts by use of transcription factors Gata4, Hand2, Tbx5, and Myocd together with miR-1 and -133. Although iCMs showed sarcomere assembly, expression of some cardiac genes, downregulation of fibroblast genes, and Ca2+ transients, the vast majority of iCMs failed to upregulate a host of cardiac genes and spontaneously beating iCMs were sparse, suggesting a partially reprogrammed state in the majority of transduced cells. Using only transcription factors, Fu et al. [7] aimed to demonstrate in vitro reprogramming of human fibroblasts to iCMs with the GMT factors. GMT alone did not result in reprogramming of human fibroblasts; however, addition of MESP1 and ESRRG to the GMT factor mix resulted in global shifts in cardiac gene-expression and changes in phenotype toward cardiac morphology [7]. Adding Myocd and ZFPM2 to this cocktail further improved reprogramming, with resultant iCMs assembling sarcomeres and exhibiting cardiac cell-like electrophysiology [7]. However, the iCMs were not observed to beat, even long after reprogramming, despite demonstrating similar global gene expression levels as were achieved by GMT reprogramming of mouse fibroblasts in vitro [7]. Wada et al. [8] also demonstrated that GMT factors, MESP1 and Myocd together

6 were capable of inducing cardiac gene expression in human fibroblasts, although the iCMs did not beat spontaneously. Interestingly, culturing these iCMs with mouse cardiomyocytes resulted in their beating [8]. While these studies demonstrate that human fibroblasts are indeed amenable to cardiac reprogramming, they also underscore the need to better understand the barriers to complete reprogramming as well as the mechanism of reprogramming. Recently, several groups have made exciting headway in these directions.

7. Challenges and Future Objectives While many exciting developments have been made in the emerging field of direct cardiac reprogramming, many challenges must be addressed to move this technology closer toward applications in disease modeling, drug screening and ultimately toward treating human heart diseases. One challenge is the current inefficiency of in vitro cardiac reprogramming in both human and mouse fibroblasts. The first reported method of in vitro reprogramming fibroblasts into iCMs by GMT cocktail demonstrated an efficiency of iCM generation at